Characterization of VCAM/ALB8 bispecific nanobody. (a) Binding of VCAM/ALB8 to VCAM-expressing cells, (b) binding of VCAM/ALB8 to human serum albumin, measured using plate-based ELISA, (c) simultaneous binding of VCAM/ALB8 to VCAM-expressing cells and to human serum albumin, measured using fluorescently labeled HSA, mouse VCAM-1 expressing cells (or controls), and varying concentrations of VCAM/ALB8, (d) whole-body biodistribution of VCAM/ALB8 and untargeted control, as compared to monovalent forms, and (e) distribution of VCAM/ALB8 and untargeted control in naive and injured animals. Comparisons were made by one-way ANOVA with Tukey’s post-hoc test, * (p < 0.05), ** (p < 0.01), *** (p < 0.001). All biodistributions were performed 20 min post-injection of 10 μg of bispecific nanobody.